Rituximab is widely used as a therapy for B cell malignancies. Despite its certain clinical value, the mechanisms responsible for the clinical anti-tumor effect of rituximab and other monoclonal antibodies (mAb) are unclear. Both antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) appear to play a role. We recently reported that the C3b component of complement can block the interaction between mAb Fc and CD16. This, in turn, limits activation of NK cells by rituximab-coated target cells and blocks NK-mediated ADCC. This finding has led to the hypothesis that complement fixation blocks mAb-induced NK activation and ADCC, and limits the efficacy of mAb therapy of B cell malignancies. If this hypothesis is correct, a number of strategies that avoid complement fixation could be used to enhance the efficacy of anti-B cell mAb therapy. These include (1) depletion of complement prior to mAb therapy, (2) use of mAb engineered to interact with CD16 on NK cells but not fix complement and (3) use of mAb at doses that activate NK cells but do not fix complement. The studies described in this proposal will further assess the validity of the underlying hypothesis, and assess the potential of these strategies. This will be done through three specific aims. Specific Aim 1 will evaluate in vitro the relationship between complement and NK activation by rituximab-coated target cells. Specific Aim 2 will use immunocompetent murine models to evaluate how (1) mAb dosing, (2) depletion of complement, and (3) the ability of mAb to fix complement, impact on the efficacy of therapy and the biologic effects of mAb at the site of the tumor. Specific Aim 3 will evaluate the effect of rituximab on complement activation, NK activation and ADCC in patient samples. Samples will be obtained at key time points from patients receiving an initial infusion of rituximab in two different clinical scenarios. The research described in this program could lead to a number of novel strategies for improving the efficacy of mAb of B cell malignancies that could be applicable to other mAb-based cancer treatments. PUBLIC HEALTH RELEVANCE: Despite the certain clinical value of monoclonal antibody therapy of cancer, there is much to learn about the mechanisms responsible for its anti-cancer activity. Preliminary data suggests that, contrary to current understanding, complement can block monoclonal antibody induced NK cell activation and antibody dependent cellular cytotoxicity, and limit the efficacy of therapy. The current proposal is designed to evaluate this hypothesis, as well as strategies designed to overcome this effect, in vitro, in animal models and in clinical correlative studies.